Yakko (voiced by Rob Paulsen) - Yakko is a loquacious, wise-cracking smart-aleck, who usually acts as the leader of the trio. He is also obsessed with girls, and is known for saying "Goodnight, everybody!" after a joke that only adult viewers will understand. Yakko wears tan pants with a blackbelt.

Wakko (voiced by Jess Harnell) - Wakko has a Liverpudlian accent, a huge appetite and a "gag bag" full of tricks. He also appears to be the least intelligent, or most insane, although Yakko has claimed it to be "middle kid syndrome" in the episode "Survey Ladies". Wakko wears a red baseball cap backwards and a light blueturtleneck.

Dot (voiced by Tress MacNeille) - Dot is cute, sassy and more easily relaxed than her brothers, but proves on numerous occasions that she can be just as zany. Her full name is "Princess Angelina Contessa Louisa Francesca Banana Fanna Bo Besca III". She despises being referred to as "Dottie", threatening "Call me Dottie and you die." She wears a pinkskirt with a flower in her ears. While no writer or creator has confirmed the fact, it has been suggested that Dot's name comes from the period, or 'dot', found at the end of 'Warner Bros.'.

Dr. Otto Scratchansniff – German-accented studio psychiatrist, voiced by Rob Paulsen, who attempts to force the Warners to be less zany. He often loses patience with the Warners and goes insane, once pulling his hair out until he was bald.[1] He becomes fonder of the Warners and takes more responsibility for them, occasionally acting as a father figure. The Warners are clearly shown to be acting crazy around him on purpose, but are also fond of him.

Hello Nurse – Buxomblonde studio nurse (who also appears in other occupations) voiced by Tress MacNeille, over whom Yakko and Wakko fawn. Her appearance usually prompts the boys into affectionate greetings ("Hellooooooo, Nurse!") followed by leaping into her arms, although sometimes they say the same thing to other characters, leaping into their arms in the same manner. Hello Nurse appears in a few Slappy cartoons as a running gag. In Wakko's Wish, it is learned that her "mean IQ (is) 192" and she laments that she is respected for her looks and not her mind.[1] The phrase was initially meant to be used as a catchphrase for Buster Bunny on Tiny Toon Adventures as a counterpart for Bugs Bunny's "Eh, what's up, doc?", however, the writers could not find an appropriate way for him to use the phrase organically. The phrase “Hellooooooo, nurse!” did not originate with Animaniacs; it had been used decades earlier in vaudeville shows.

Pinky and the Brain are an imbecilic white mouse and his genius companion, voiced by Rob Paulsen and Maurice LaMarche, respectively. The Brain is serious and clearly the leader, and attempts to take over the world; Pinky is eccentric and unintelligent, but loyal to the Brain. In 1995, they were spun off into a cartoon series of their own.

Walter Wolf – Slappy Squirrel's longtime nemesis, voiced by Frank Welker in his first appearance and Jess Harnell for the remainder of the series; is a parody of the Big Bad Wolf characters of Disney and Tex Avery. In "...And Justice For Slappy", he has an adult grandson.

Sid the Squid – Villain, voiced by Jack Burns, who appeared in five Slappy cartoons: "Hurray for Slappy", "Scare Happy Slappy", "Rest in Pieces", "Macadamia Nut", and 'Star Warners". He is first mentioned in some episodes of Batman: The Animated Series. (Note: this character has no association with the Slappy "villain")

Beanie the Brain-Dead Bison – Villain similar to Pete Puma, voiced by Avery Schreiber, who appeared in "Hurray for Slappy", "Scare Happy Slappy", "Rest in Pieces", "Macadamia Nut", and "Star Warners".

Candie Chipmunk – Slappy's self-centred neighbour, voiced by Gail Matthius, who appears in "I Got Yer Can". An excerpt of the "Dance of the Reed Flutes", from Tchaikovsky'sNutcracker Suite, plays when she appears.

The Godpigeon – A pigeon who is an unintelligible parody of Marlon Brando's Don Vito Corleone in The Godfather. The bird's bulky appearance signifies Brando's massive weight gain, due to overeating habits in his later years. Bobby usually acts as interpreter for his gibberish.

The Girlfeathers are their girlfriends. Sasha, voiced by Tress McNeille, is Squit's girlfriend and Pesto's sister. Lana (voiced by Gail Matthius) is Bobby's girlfriend, a parody of Cathy Moriarty's character in Raging Bull. Kiki, Pesto's girlfriend, is a stereotypical dumb blonde.

Rita and Runt are escaped stray pets, usually searching for a home. In the end, they are either rejected or threatened and back as strays. While Rita (voiced by Bernadette Peters, with vocal effects by Frank Welker) is a smart and often sarcastic singing cat, Runt (voiced by Welker) is a very loyal but dumb dog who thinks that Rita is also a dog like himself and who constantly uses the word, "definitely" a lot when speaking (for example, "Rita, you're a good dog. Definitely a good dog!").

These segments, along with "Minerva Mink", were discontinued at the end of Season 1 (in part, because of Bernadette Peters' salary[citation needed]). Welker remained a series regular, voicing other characters.

Buttons is a Rough Collie who takes care of Mindy and is voiced by Frank Welker. Mindy, voiced by Nancy Cartwright, is a young girl who is constantly getting into trouble and says "OK, I love you, bye-bye." after she says "why" to someone that she does not know, even to Buttons. Buttons knocks himself out rescuing Mindy, only to be blamed and punished for Mindy's misbehavior. There is no consistency or continuity: in one episode, the family and Buttons are costumed superheroes. In another they are mer-people (fish tails instead of legs), and one episode was set in a prehistoric age.

Newt – Faithful dachshund of his lazy owner, who also appears in "Puttin' on the Blitz" with Rita and Runt as "Schnappsie". Voiced by Arte Johnson (Newt) and by Frank Welker (Schnappsie).

Wilford B. Wolf – Nerdy wolf who becomes a handsome werewolf every full moon, voiced by Peter Scolari. His werewolf alter-ego is built like a Chippendales dancer and dresses like one, and is very affectionate to Minerva. Minerva once asked him about his werewolf changes. Unfortunately, he was in his nerd alter-ego and launched into a long, boring explanation replete with mathematical diagrams and charts.

Chicken Boo – Six-foot-tall chicken, voiced by Frank Welker. He wishes to live as a human, so he wears flimsy disguises, usually just a hat or a coat, which somehow always fool everyone (except for one person who no one believes). Unlike other animal characters, Boo cannot talk and acts almost exactly like a real chicken, making his disguises all the more absurd. At the end of each episode, his disguise falls apart, and he is exposed as a giant chicken. This causes all of his previous supporters to turn against him, usually running him out of town.

Steven Spielberg (voiced by Frank Welker) – The show's executive producer, often mentioned by the cast.

Colin (the Randy Beaman Kid) – Wide-eyed boy who tells improbable stories which allegedly happened to his (never-seen) friend, Randy Beaman. He comes out of his home with an object in hand that he fidgets with while he tells the story (the object is usually not related to the story) and starts off with "One time...okay, see, one time..." and then tells his story while playing with whatever he has. When he is finished, something will usually happen with the object that he is playing with (like an ice cream cone melting or a baseball going through someone's window), and the kid will finish with "'kay, bye." and walk back into his house.

The Flame – Childlike candle flame, voiced by Luke Ruegger, who is present at important historical events and teaches fire safety.

Flavio and Marita – Also known as the Hip Hippos, a wealthy, Spanish hippo couple voiced by Frank Welker and Tress MacNeille.

Dr. Jane Embryo – Zoologist who studies the Hip Hippos and tries in vain to protect them, a parody of Joan Embery and Jane Goodall voiced by Tress MacNeille.

Mr. Cory Skullhead – Muteskeleton seen in the "Good Idea-Bad Idea" segment narrated by Tom Bodett and a parody of Edward Scissorhands His first appearance is in "Nighty-Night Toon", where he scares Buttons under the bed.

The Mime – Nameless mime who appears in "Mime Time", also narrated by Bodett.

Katie Ka-Boom – A teenage girl voiced by Laura Mooney,[4] who morphs into various violent, destructive monsters when things do not go her way. She lives with her parents and her little brother named Tinker. Katie is the only member of her family who has blonde hair while the rest of her family are brunettes. The premiere Katie Ka-boom segment was also a Chicken Boo crossover, in which Katie morphs into a green Incredible Hulk-like monster when initially told her new boyfriend (who is also the student council president, class valedictorian and the school's star wrestler) is a giant chicken, and then morphs into a monster made of fire when she herself realizes that he is a giant chicken and ends their relationship.

Mary Hartless – Parody of Mary Hart, voiced by Valri Bromfield and Tress MacNeille, who appears as a newsreader with a variety of hairstyles in "Hurray for Slappy", "Chairman of the Bored", "Bubba Bo Bob Brain", and "Critical Condition".

The Narrator – Offscreen narrator (voiced by Jim Cummings) whose voice resembles Cummings' voice for Winnie the Pooh, who appears in "Nighty-Night Toon", "Gift of Gold", and "Warners and the Beanstalk".

Francis "Pip" Pumphandle (voiced by Ben Stein) a man who tells long and boring stories, and bores the Warners in "Chairman of the Bored". He also appears in the Pinky and the Brain episode "Star Warners", and returns in Wakko's Wish as a Desire Fulfillment Facilitator.